The human bladder is the most sensitive internal organ to arsenic-induced carcinogenesis. The bladder is exposed to inorganic arsenicals and methylated metabolites both systemically and via the urine. Methylated metabolites of arsenic have been found to be more cytotoxic than inorganic arsenicals. The UROtsa cell, an immortalized human urothelium cell, has become an accepted system for examining the toxic effects of arsenicals to the bladder. Year-long chronic low-level exposure of arsenite [1 microM As(III)] or the more toxic arsenic metabolite [monomethyl-arsonous acid;50 nM MMA(III)] transformed UROtsa cells such that they formed tumors when injected into immuno-deficient mice. Current studies indicate that much shorter exposures (3 months) to low-level MMA(III) is sufficient to allow them to grown in soft agar and form tumors in immuno-deficient mice. These results indicate that MMA (III) causes critical, irreversible events in UROtsa cells in the first 3 months of exposure. Thus the Goal of this proposal is to find the critical events that short-term, low-level As(III) or MMA(III) causes in UROtsa cells resulting in their eventual transition into malignantly transformed cells. To accomplish this goal we will do low-level, short-term (0-3) exposures of UROtsa cells to arsenicals [1 microM As(III) and 50 nM MMA(III)] to determine the minimal exposure and time required to malignantly transform these cells. Once this minimal exposure time point is established we will examine the cells for changes in specific alterations in macromolecules, signaling systems, or regulatory systems resulting in the irreversible changes. Since previous studies have found reactive oxygen species (ROS) generation by low-level exposure to As(III) and MMA(III), we will determine if these early effects of arsenicals on UROtsa cells are mediated by ROS generation. To determine if low-level arsenicals can transform "normal" human bladder cells, primary cultures of human bladder cells will be similarly examined. Lastly pivotal events in the transformation of bladder cells by low-level arsenicals will be examined as possible biomarkers in exposed populations. Overall, these studies will clarify the toxic effects of low-level arsenic in a human bladder model and provide potential biomarkers for arsenic-induced bladder injury.